Effect of different water contents in the substrate on cultivation of Pleurotus ostreatus Jacq. P. Kumm

White-rot fungi comprise a large group of organisms known for their ability to produce different types of enzymes for the degradation of lignin and cellulose. Lignin is the main component of wood and other plant biomass, and, together with cellulose and hemicellulose, it is the main source of carbon for the mycelium (Baldrian et al., 2005). One representative of the white-rot fungi is Pleurotus ostreatus, which produces ligninolytic and hydrolytic enzymes, such as laccase (EC 1.10.3.2), Mn-dependent peroxidase (EC 1.11.1.13), 1,4-β-glucosidase (EC 3.2.1.21), cellobiohydrolase (EC 3.2.1.91) and many more for the degradation of lignocellulosic substrates (Elisashvili et al., 2001; Mikiashvili et al., 2004, 2006).

Several studies have investigated the relationships among specific enzyme activity and lignocellulose degradation, biological efficiency (BE) and other processes. During cultivation of Pleurotus spp., the activities of different enzymes change. For example, the activity of 1,4-β-glucosidase and cellobiohydrolase increases during cultivation, which may be due to the decomposition of cellulose in the later stages of fungal growth (Wang et al., 2001; Baldrian et al., 2005). The activity of 1,4-β-glucosidase is also affected by other parameters such as the pH of the medium, the temperature and the humidity of the substrate (Goyal and Soni, 2011). Another enzyme involved in the degradation of cellulose is cellobiohydrolase. When cellulose is degraded, the enzymes behave synergistically and produce carbohydrates, which can be easily assimilated by fungal mycelia. The highest activity of cellobiohydrolase was recorded in the phase of primordial deployment and fructification (Velázquez-Cedeño et al., 2002). Manganese-dependent peroxidase (Mn-P) and laccase play an important role in lignin decomposition (Maciel et al., 2012). The first is an extracellular haeme protein containing a carbonyl group and requiring H₂O₂ catalysis with the involvement of Mn⁺; Mn-P is secreted during mycelium growth and catalyses oxidation, resulting in the formation of phenoxy radicals. Subsequently, a series of non-enzymatic catalysed reactions and further degradation follow (Jin et al., 2021). Laccase is a copper-containing oxidase and can deprotonate hydroxyl groups to form phenoxy anions, leading to the breakage of the side chains of lignin-type structural units (Ihssen et al., 2017). The activity of lignin-degrading enzymes decreases during cultivation (Kannan et al., 1990), and the production of these enzymes depends on the culture medium, pH, temperature and the growth phase of the fungus (Isikhuemhen and Mikiashvilli, 2009; Knop et al., 2015).

An important criterion for the cultivation of P. ostreatus is the water content of the substrate as it impacts mycelium growth, yield and enzymatic activities. The optimum water content for P. ostreatus cultivation is between 65% and 75%. A high initial water content can cause misshaped fruiting bodies (Wang et al., 2001; Siwulski et al., 2007; Dos Santos Bazanella et al., 2013). Although a high initial moisture level is the best condition for laccase activity, for Mn-peroxidase activity, a low initial moisture level is more suitable (Dos Santos Bazanella et al., 2013).

However, the relationships among enzyme activity, substrate moisture and different substrate layers are still largely unclear. This study is the novel comparison of the activities of multiple enzymes (Mn-dependent peroxidase, 1,4-β-glucosidase and cellobiohydrolase) produced by P. ostreatus and their relationships with other parameters, such as pH, water content within different substrate layers at different initial water content of substrate and its impact on mycelial growth and BE of the substrate.

Based on the results in Table 1, the water content of the substrate gradually decreased, which was most pronounced in the variant with 60% moisture (from 59.4% to 41.4%). During the colonisation phase, in most variants, higher water content values were measured in the upper layer of the substrate, whereas during fructification (from the primordia phase to the harvest phase), a higher water content value was found in the centre of the substrate. The lower water content in the upper layer of the substrate can be explained by the formation of primordia and their growth, which uses water from the substrate and transports it to the atmosphere. This finding is in agreement with the results of Atila (2019) for the cultivation of Hericium erinaceus. In this experiment, the author used different substrates such as oak sawdust, wheat straw or poplar sawdust with initial moisture levels of 68.5%–70%. During cultivation, the water content decreased by up to 17.3%. In our experiment, significant differences were found between the layers in the individual phases of cultivation, with the exception of the variant with a substrate water content of 70% and decrease was up to 27%. Furthermore, significant differences in water content values were found during the various stages of cultivation, especially between the colonisation of the substrate and the harvesting of the fruiting bodies. Bellettini et al. (2019) reported that water content in the substrate higher than 70% could cause development of diseases. However, in our case, the development of diseases and mould did not occur, even at a water content of 75%.

Another studied parameter of the substrate was pH. Based on the results, the pH value decreased during colonisation from 6 (before inoculation) to 4.74–5.43. This finding is in agreement with Bellettini et al. (2019), who reported that substrate pH is reduced, which is close to 4, during the colonisation stage. Obodai et al. (2003) used composted sawdust, banana leaves, maize stove and rice straw as substrates for cultivation, with initial pH values of 6.92–7.6 at a water content of 58%–70%. The pH values were higher than those observed in our study, most likely because of the different substrate composition. In our experiment, the pH value slightly increased from the colonization phase for all variants during cultivation. The lowest pH value (4.74) was measured for the variant of 60% water content in the substrate in the upper layer in the colonisation phase, and the highest pH value was found for the variant of 75% water content in the substrate in the harvest phase (5.83). In the colonisation phase, the pH values of all variants were higher in the middle layer of the substrate. In the primordial deployment phase, the pH of the upper layer was higher in the variant with 60% water content in the substrate; in the 65% and 70% water content variants, the pH was higher in the centre of the substrate. At the harvest stage, the pH in the centre layer was higher only for the 75% water content in the substrate. There was a statistically significant difference in pH values between the individual substrate layers, with the exception of the 70% water content in substrates, where no difference in the pH values was found, even in the individual phases of cultivation. For the 60%, 65% and 75% variants, statistically significant differences were found between the individual cultivation phases, especially between the colonisation and harvesting phases (Table 1).

The mycelium growth rates of P. ostreatus are presented in Figure 1. Variants with water contents of 65% and 75% showed the highest increase in mycelium growth per day. Between these two variants, there were no statistically significant differences. On the other hand, statistically significant differences were found between variants 60% and 70%, which also differed from the 65% and 75% variants. The variant with the lowest water content in the substrate had the lowest mycelium growth, which leads us to infer that an initial substrate water content of 60% is insufficient for adequate mycelium growth. Zervakis et al. (2001) tested growth rate of P. ostreatus and other mushrooms on different substrates with 65%–70% water content. Growth on wheat straw was 6.2 mm × day⁻¹ which was slightly higher than that in our experiment (5.26 mm × day⁻¹). However, growth rate in our experiment was higher than that of P. eryngii, P. pulmonarius or Lentinus edodes, tested in their experiment.

Figure 1.

The BE is the ratio of the weight of the fresh fruiting body (g) per dry weight of substrate (g), expressed as a percentage (Liang et al., 2019). In our experiment (Figure 2), we found no statistically significant differences among the variants. However, the highest BE values (50% and 51%) were found for variants with 65% and 70% water content in the substrate, respectively. On the other hand, the lowest BE was found for the variant with 60% water content in the substrate, along with the lowest mycelium growth, indicating that this level of substrate moisture is not suitable for both adequate mycelium growth and high yields. In the experiment of Kalmıs et al. (2008), P. ostreatus was cultivated on wheat straw mixed with olive mill effluent, and BE ranged from 14.7% to 50.7% at 70% water content in the substrate. Compared with our study, BE was lower for all substrate mixes Girmay et al. (2016) conducted experiments with different substrates. and BE of P. ostreatus cultivated on wheat straw (60%–70% water content in substrate) was 35.88%, which is higher than that in our experiment at a 60% water content and lower than our value for 65% and 70%.

Figure 2.

The highest 1,4-ß-D-glucosidase activity (Table 2) was measured in the variant with a water content of 75% in the substrate at the harvest of fruiting bodies, when the activity increased over time in both substrate layers. Kannan et al. (1990) also found that the activity of 1.4-ß-D-glucosidase during the cultivation of Pleurotus sajor-caju increased over time. In their experiment, papermill substrate was used, with a cultivation period of 30 days. In our experiment, a statistically significant difference was found in this variant between the colonisation and primordial deployment phases and the harvest phase. This trend was also found for cellobiohydrolase activity at a water content of 75% in both layers, and at the same time, the highest values of cellobiohydrolase activity were measured (3,349.13 and 4,083.42 μmol · hr⁻¹ · g⁻¹ respectively). The values of 1,4-ß-D-glucosidase and cellobiohydrolase showed a strong correlation (correlation coefficient = 0.97), and the activities of these enzymes also correlated with the pH (correlation coefficients: 0.55 and 0.49). In the experiment of Velázquez-Cedeño et al. (2002), activity of cellobiohydrolase also increased during cultivation and was at its highest at the fruiting stage. The Mn-peroxidase values decreased during cultivation, and a negative correlation (-0.24 and -0.25) was found in connection with 1,4-ß-D-glucosidase and cellobiohydrolase (Table 2). This result is consistent with the findings of De Souza et al. (2006) who noted an increase in peroxidase activity within 10 days, followed by a decrease until 30 days of cultivation. Kurt and Buyukalaca (2010) cultivated P. ostreatus on different agricultural wastes. They reported the highest laccase activity on 10th day of cultivation, and then, activity slowly decreased. In our experiment, the laccase activities were below the detection limit.

The substrate moisture content decreased during cultivation, which can be attributed to development of P. ostreatus culture. The upper layer had a higher water content than the center of the substrate during primordia development and fructification because of the transport of water to the surface of the substrate for the growth of the fruiting bodies. The optimum substrate water content for mycelium growth and BE was 65%–75%, similar to previous studies. A lower water content is not suitable for proper mycelium growth and good yield. The activities of hydrolytic enzymes (1,4-ß-D-glucosidase and cellobiohydrolase) increased during cultivation, which is connected to the gradual cellulose degradation. Variants with higher water contents in the substrate showed greater activities of these enzymes, which were correlated with the pH. The enzyme Mn–P is involved in lignin degradation, and its activity was higher in the early stage of cultivation. Differences in the activities in different substrate layers were significant mostly for variants with 65% and 70% water content, which also had the highest BE values. Consequently, for the cultivation of P. ostreatus, the most suitable substrate water contents range between 65% and 70%.